The present invention relates to a method for the synthesis of peptides without any solvent.
Peptides are presently considered as pharmaceutical active ingredients because of their high therapeutic index and their low toxicity. Because of the development of novel administration systems which increase their bioavailability, it is expected that the market of therapeutic peptides will rapidly develop during future years. But there always exists a need for effective methods for synthesizing these compounds. In spite of well-established production procedures (in solution, in a solid phase, by recombination), there still subsists many development problems associated with the enormous amount of solvent required for the synthesis, notably on solid supports (2,000-5,000 kg for a large peptide).
The field of “green chemistry” is becoming more extensive because of the seriousness of present environmental problems. Several fields of this subject have emerged: the use of an alternative raw material and of a non-toxic reagent, use of natural processes, use of alternative solvents, design of safer chemical products, development of other reaction conditions, minimization of energy consumption . . . . A particular active field lies in the use of alternative solvents such as aqueous, ionic, fluorinated or supercritical liquids, in order to replace volatile organic or chlorinated solvents and in order to solve the problems of treating or recycling solvent-based waste.
An alternative approach consists of producing chemical reactions in the absence of a solvent. Techniques such as mixing or milling of solids have proved to be efficient. Nevertheless, no application of these techniques to fields such as peptide or amino acid synthesis has been undertaken, in spite of the importance of these biomolecules.
The authors of the present invention have discovered surprisingly novel routes for synthesizing peptides under conditions without any solvent. Surprisingly, the authors have achieved the coupling of carboxy anhydrides of amino acids protected by a urethane (Urethane-protected N-CarboxyAnhydride (UNCA)), with amino acids or amino esters, while all these compounds remained in their solid form, under conditions of ball milling and at room temperature.
More specifically, the invention relates to a method for synthesizing a compound of the following formula (I)
wherein:
n is an integer larger than or equal to 1, advantageously comprised between 1 and 100, more advantageously comprised between 1 and 50, still more advantageously equal to 1 or 2;
Rb and each Rn represent independently of each other a hydrogen atom, an aryl (C1-C6 alkyl) group, or a C1-C6 alkyl group either substituted or not by an aryl group, —COOH, —COO—(C1-C6 alkyl), —CONH2, —SH, heteroaryl, —NH2, —NHC(NH)(NH2), —S—(C1-C6 alkyl)-OH or phenol group, the groups of which —COOH, —NH2, OH, SH and NH are optionally protected with one or more identical or different N-protective or O-protective groups and different from Ra, advantageously these N-protective and/or O-protective group(s) are stable under conditions for removing the Ra group;
Ra represents an N-protective group;
Rc represents an —ORd group wherein Rd represents a C1-C6 alkyl group or an —NReRf group wherein Re and Rf represent independently of each other an N-protective group,
characterized in that it comprises a step (a) consisting of reacting in the presence of a base and without any solvent, the compound of the following formula (II):
wherein Ra and Rb are as defined earlier, with the compound of the following formula (III):
wherein n, Rn and Rc are as defined earlier, as well as its pharmaceutically acceptable salts, preferably chlorides, acetates and trifluoroacetates.
In the sense of the present invention, by the term “without any solvent” is meant that the reaction occurs in the absence of a solvent. A solvent according to the invention is a product which solubilizes the reagents but does not directly participate in the reaction. Thus, within the scope of the present invention, the base is not a solvent.
Moreover, in the method according to the invention, all the reagents used are in the solid state. This is in particular the case of the compounds of formula II and III and of the base. Thus, this reaction occurs in the solid state and not in solution. Advantageously, these reagents are in a finely divided solid form, as obtained by ball-milling. The advantage of this type of reaction is to suppress the use of a solvent (green chemistry) but also to facilitate application of the reaction, treatment and to allow very pure products to be obtained.
By the term of “C1-C6 alkyl group” is meant in the sense of the present invention any linear or branched alkyl group with 1-6 carbon atoms, in particular the methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl groups. Advantageously this is a methyl or t-butyl group. By the term of “aryl group” is meant in the sense of the present invention one or more aromatic rings having 5-8 carbon atoms, which may be placed side by side or fused. In particular, aryl groups may be monocyclic or bicyclic groups, preferably a phenyl, napthyl, tetrahydronaphthyl or indanyl group. Advantageously, this is a phenyl group.
By the term of “heteroaryl group”, is meant in the sense of the present invention any hydrocarbon aromatic group with 3-9 atoms, containing one or two heteroatoms, such as for example sulfur, nitrogen or oxygen atoms. The heteroaryl group according to the present invention may be formed with one or two fused cycles or cycles placed side by side. Examples of heteroaryl groups are the furyl, isoxazyl, pyridyl, pyrimidyl, benzimidazole, benzoxazole, benzothiazole groups.
By the term of “N-protective group” is meant in the sense of the present invention any substituent which protects the NH2 group against undesirable reactions such as the N-protective groups described in Greene, “Protective Groups In Organic Synthesis”, (John Wiley & Sons, New York (1981)) and Harrison et al. “Compendium of Synthetic Organic Methods”, Vols. 1-8 (J. Wiley & Sons, 1971-1996). The N-protective groups comprise carbamates, amides, N-alkylated derivatives, amino acetal derivatives, N-benzylated derivatives, imine derivatives, enamine derivatives and N-heteroatom derivatives. In particular, the N-protective group comprises the formyl, acetyl, benzoyl, pivaloyl, phenylsulfonyl, benzyl (Bn), t-butyloxycarbonyl (boc), benzyloxycarbonyl (cbz), p-methoxybenzyloxycarbonyl, p-nitrobenzyl-oxycarbonyl, trichloroethoxycarbonyl (troc), allyloxycarbonyl (alloc), 9-fluorenylmethyloxycarbonyl (fmoc), trifluoroacetyl group, benzyl carbamates (either substituted or not) and the like. Either boc or cbz as a N-protective group is advantageous to use because of the relative facility of removal, for example with moderate acids in the case of boc, for example trifluoroacetic acid, or hydrochloric acid in ethyl acetate; or by catalytic hydrogenation in the case of cbz. Advantageously, this is the boc group.
By the term of “O-protective group” is meant in the sense of the present invention any substituent which protects the hydroxyl or carboxyl group, i.e. a reactive oxygen atom, against undesirable reactions, such as the O-protective groups described in Greene, “Protective Groups In Organic Synthesis”, (John Wiley & Sons, New York (1981)) and Harrison et al. “Compendium of Synthetic Organic Methods”, Vols. 1-8 (J. Wiley & Sons, 1971-1996). The O-protective groups comprise methyl or alkyl ethers either substituted or not, for example methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, t-butyl, benzyl and triphenylmethyl, benzyl ethers (either substituted or not), tetrahydropyranyl ethers, allyl ethers, substituted ethyl, for example 2,2,2-trichloroethyl, ethers, silyl ethers or alkylsilyl ethers, for example trimethylsilyl, t-butyl dimethyl silyl, and t-butyldiphenyl silyl, heterocyclic ethers; and esters prepared by reaction of the hydroxyl group with a carboxylic acid, for example tert-butyl, benzyl, or methyl esters, carbonates in particular benzyl carbonate or haloalkyl carbonate, acetate, propionate, benzoate and the like. Advantageously this is the benzyl group.
Subsequently, the compounds of formula I may be deprotected so as to obtain the peptides for which the —OH, —NH2, —SH, —NH and —COOH functions are non-protected.
In an advantageous embodiment of the invention, n is equal to 1 and step (a) consists of reacting in the presence of a base and without any solvent the compound of formula (II) with the compound of the following formula (III-1):
wherein R1 and Rc are as defined earlier so as to obtain the compound of the following formula (I-1):
wherein Ra, Rb, Rc and Rn are as defined earlier.
Step (a) is advantageously carried out by means of ball-milling. It is particularly advantageous to carry out the step (a) from freshly prepared starting products of formula (II) and (III). The base is a solid base and may advantageously be selected from the group formed by carbonates, in particular sodium, potassium and cesium hydrogencarbonates, and sodium, potassium and cesium carbonates. Advantageously this is sodium hydrogencarbonate.
The substituents Rb and each substituent Rn may independently be selected from the group consisting of hydrogen, —CH3, the benzyl group, —CH2CONH2, —CHCH3C2H5, —(CH2)3NHC(NH)(NH2), —CH(OH)CH3, —CH2COOH, —CH2SH, —CH2CH2COOH, —CH2CH2CONH2, imidazolyl methyl, propyl, —CH2CH(CH3)2, —(CH2)4NH2, —(CH2)2SCH3, —CH2OH, indol-2-ylmethyl, p-methylphenol, isopropyl groups. As indicated earlier, the NH2, NH, COOH, SH and OH function of these groups are advantageously protected by one or more identical or different, O-protective and/or N-protective groups and different from Ra. Advantageously, these N-protective and/or O-protective group(s) are stable under the conditions for removing the Ra group.
In an advantageous embodiment of the invention:
Ra, Re and Rf are selected independently of each other from the group consisting of tert-butyloxy carbonyl, 9-fluorenylmethyloxycarbonyl, benzyloxycarbonyl, nitro-veratryloxy carbonyl and/or
Rb is selected from the group consisting of isopropyl, benzyl and —CH2COOt-Bu and/or
each Rn is selected independently of the others from the group consisting of methyl, benzyl and —CH2CH(CH3)2 and/or
Rd is selected from the group consisting of methyl and tertiobutyl.
In an advantageous embodiment of the invention, the compound (II) is selected from the group consisting of the compounds of the following formulae (II-a), (II-b) and (II-c):

the compound (III) is selected from the group consisting of the compounds of the following formulae (III-a), (III-b), (III-c), (III-d) and (III-e):

In another advantageous embodiment of the invention,
the compound of formula (II) has the following formula (II-2):
wherein Ra is as defined earlier,
the compound of formula (III) has the following formula (III-2):

and the compound of formula (I) has the following formula (I-2):
wherein Ra is as defined earlier.
The compound of formula (II-2) may be prepared by a method comprising the following successive steps:
1) protecting the amine group of the compound of the following formula (IV):
with an N-protective group Ra in order to form the compound of the following formula (V):

2) reacting the compound of formula (V) with a benzyl halide in the presence of a base, advantageously cesium carbonate, in order to obtain the compound of the following formula (VI):
wherein Ra is as defined above;
3) protecting the amine group of the compound of formula (VI) with an N-protective group Rg in order to form the compound of the following formula (VII):
wherein Ra and Rg are as defined above;
4) reducing the compound of formula (VII) into a compound of the following formula (VIII):
wherein Ra and Rg are as defined above;
5) cyclizing the compound of formula (VIII) into a compound of formula (II-2) by reaction with DMF and oxalyl chloride. The substituents Ra and Rg advantageously represent tert-butyloxycarbonyl.
In an advantageous embodiment, the method comprises the following additional successive steps:                (b) reacting the compound of formula (I-2) obtained in step (a) with an acid, advantageously hydrochloric acid gas, in order to form the compound of the following formula (I-3):        

(c) reacting the compound of formula (I-3) with a base, advantageously sodium bicarbonate, in order to form the compound of the following formula (I-4):
